Abnormal fetal growth increases the risk for perinatal complications and predisposes for adult disease. Fetal growth is strongly dependent on nutrient availability, which is determined by placental nutrient transfer. The activity of key placental amino acid transporters is decreased in intrauterine growth restriction (IUGR) and up- regulated in fetal overgrowth, suggesting that changes in the activity of placental nutrient transporters directly contribute to abnormal fetal growth. However, mechanistic information on the regulation of placental nutrient transporters is currently lacking. We recently reported that mammalian target of rapamycin (mTOR) signaling constitutes a key regulator of trophoblast amino acid transporters;however the underlying molecular mechanisms are unknown. Central hypothesis: Both mTOR Complex 1 (mTORC1) and 2 (mTORC2) regulate placental amino acid transporter activity by affecting the plasma membrane trafficking of transporters. We further propose that the molecular mechanisms involved are distinct in that mTORC1 activation phosphorylates the E3 ubiquitin ligase Nedd4-2, which decreases transporter ubiquitination resulting in increased amino acid transporter expression at the cell surface whereas mTORC 2 activation stimulates the actin skeleton mediated by PKC1. Specific Aims : (1) Determine the role of mTORC1 and 2 in regulating placental amino acid transporter activity, (2) Establish the effect of mTOR signaling on trophoblast amino acid transporter trafficking, (3) Identify the mechanisms by which mTOR regulates plasma membrane trafficking and activity of trophoblast amino acid transporters and (4) Determine the activity of the signaling pathway linking mTOR to amino acid transporter trafficking in IUGR and fetal overgrowth. Approach: To study cultured human primary trophoblast cells and measure the activity of System A and System L amino acid transporters and glucose transporters, and determine the cellular distribution of transporter isoforms using fluorescence imaging, subcellular fractionation and protein expression studies. Activation of specific signaling pathways will be determined by measurement of the expression of phosphorylated proteins. Using siRNA mediated silencing we will experimentally manipulate mTORC1 and mTORC2 signaling pathways and directly determine the mechanistic roles for signaling molecules in mediating the effects of mTOR on nutrient transporter trafficking and activity. In addition, these signaling pathways as well as nutrient transporter activity and trafficking will be determined in placentas from pregnancies with normal fetal growth, IUGR and fetal overgrowth. Significance: This work addresses a major gap in knowledge and will lead to the identification of key molecular mechanisms regulating placental nutrient transport and fetal growth, which will increase our understanding of how important pregnancy complications develop. Innovation: We will explore molecular mechanisms linking mTOR and nutrient transporters that have not been demonstrated previously in any mammalian tissue. Furthermore, we propose a novel model for the regulation of amino acid transporters in the human placenta.

Public Health Relevance

Abnormal fetal growth affects 15% of all babies and increases the risk for injuries at delivery and to develop obesity, diabetes, and cardiovascular disease in childhood and later in life. Altered placental nutrient transport is believed to directly contribute to changes in fetal growth and in order to better understand the underlying causes of these conditions we will identify key mechanisms regulating the transfer of nutrients in the human placenta. This research may help design novel treatments to alleviate abnormal fetal growth.